This invention relates to methods and reagents for the assay, detection, quantification, location or analysis of each of a plurality of substances of interest ("analytes") in a sample in which each substance is linked ("labelled") with another molecule or molecules capable of taking part in a chemiluminescent reaction.
For the purposes of this specification, a chemiluminescent reaction is defined as one which involves a chemical reaction that results in the emission of electro-magnetic radiation. This luminescence is to be distinguished clearly from fluorescence and phosphorescence. Here, luminescence, or more precisely, chemiluminescence also encompasses light emission from biological reactions (bioluminescent reactions).
A luminescent reaction is normally one between at least two molecules (S and L) with or without other reagents, cofactors, or a catalyst (D) or under the influence of a physical trigger. L is the substance which generates light, such as luminol. S is the substance which reacts with L to cause excitation, for example oxygen or hydrogen peroxide. D (if present) is a colactor, and/or catalyst or trigger such as an enzyme, a luciferass, or potassium ferricyanide. The reaction between L and S results in the conversion of L to an excited molecule L* and the return of this excited molecule to a non-excited state results in the emission of a photon. The reaction between L and S and the decay of L* to the non-excited state may take place spontaneously or may require the presence of the cofactor or catalyst D, or a physical trigger such as temperature. An example of such a reaction is the oxidation by H.sub.2 O.sub.2 of luminol. The catalyst and cofactors are often inorganic compounds as here, but may also be extracted from biological material such as the enzyme peroxidase which catalyses the luminescent reaction involving luminol.
These methods and reagents discussed above may be used in a wide variety of techniques such as immuno-assays, protein binding assays, nucleic acid hybridisation assays, cellular receptor binding assays and other analogous techniques which involve binding of the substance of interest with a specific binding partner or reagent. These types of linking are referred to herein as "binding or otherwise linking with".
The substances of interest may be peptides, proteins, polypeptides, nuCleic acids and other substances of biological interest.
Binding assays have been used for many years in the quantitation of molecules of biological interest. Numerous examples have been described in which the binding step is an immunological reaction, a protein binding reaction, reaction with a cellular receptor or a complementary nucleic acid hybridization reaction. Sensitive assays based on these reactions require the use of a label which can be attached or incorporated into one of the binding partners of such a reaction such that the degree of binding and hence the concentration or mass of another component of the reaction--the substance of interest--can be determined. Many variations of the basic binding reactions have been described and many different labels used, including radioisotopes, enzymes, fluorescent molecules and chemiluminescent molecules.
Various combinations of these have been used in sequence for the detection and quantitation of a wide variety of analytes ranging from small molecules such as hormones and drugs to large molecules such as nucleotides.
Generally speaking, these techniques have only been applied to the investigation of a single analyte in one test reaction, but there have been a limited number of examples where two analytes have been determined essentially using a single test procedure. The best known of these have been simultaneous immunoassays and/or protein binding assays for vitamin B12 and folic acid and also for thyroxine and thyro-trophin. In these cases the two different reactions are monitored independently using a different radioactive isotope for each. Here use is made of cobalt-57 and iodine-125 whose radioactive emissions are distinguishable using an appropriate gamma counter. Similar strategies have also been used for the simultaneous determination of lutrophin and follitrophin.
Radioactive reagents have three major disadvantages. Firstly, the method of labelling involves the use of highly radioactive and hence potentially hazardous reagents. Secondly, the shelf life of the radioactively labelled substance is often relatively short not only because by its very nature the radioactive isotope is continuously decaying, but also radioactively labelled proteins are often unstable. Thirdly, it is often difficult to label proteins sufficiently to provide a sensitively and rapidly detectable reagent. The measurement of luminesence is both highly sensitive and very rapid, the time of measurement being of the order of seconds rather than the several minutes normally required for measurement of radioactivity. The attachment either covalently or non-covalently, to substances not normally capable of taking part in a luminesescent reaction of a substance which is capable of taking part in a luminescent reaction provides a reagent which can be rapidly measured in very small quantities.
Work has been described relating to the use of different fluorescent molecules in so-called "dual labelling" systems. However, fluorescent labelling systems are usually capable of only gross analysis of substances and are not generally suitable for sensitive analysis. Also, with fluroescent systems, the sample is illuminated by U.V. radiation to measure the fluoresence and this may cause major problems due to photobleaching. Multiple analyte immunoassays based on the use of fluorophores have been described in which the different labels used have been chelates of different lanthanide metals emitting at different wavelengths. Limitations arise here because certain of the fluorophores used have low quantum yields and generally all assays based on these materials require complex instrumentation and chemistries in order to achieve the high levels of performance which are characteristic of many chemiluminescent systems (Ref.1).